Dual-luciferase reporter assays were performed to detect the transcription factors that bind to regions in the HOXA11-AS promoter. Truncated fragments of the HOXA11-AS promoter were amplified and inserted into a pGL3-Basic luciferase reporter (Promega, Madison, WI, USA) by using the restriction enzymes Mlu1 I and XhoI (TaKaRa, Japan); they were then ligated by use of T4 DNA ligase (TaKaRa, Japan) for subsequent transfection into WiT49 cells. Cultured cells that were 80%–90% confluent were transfected using a Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 48 h of transfection, firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System (Promega). Relative luciferase activity was determined by using Renilla luciferase activity as an internal control.
Next, dual-luciferase reporter assays were performed to confirm the binding of HIF-1α, HOXA11-AS, C/EBPβ, and HOXA11-AS. First, the binding sequences for HIF-1α or C/EBPβ on HOXA11-AS were synthesized (WT) and mutated (Mut); after which, pGL3-promoter vectors (Thermo Fisher Scientific) containing the binding sites were constructed by GenePharma (Shanghai, China). Next, the vectors were transfected into WiT49 cells treated with control conditions or hypoxia. Finally, a Dual-Luciferase Reporter Assay System (Promega) was used to detect luciferase activity.
Next, dual-luciferase reporter assays were performed to confirm the binding of HIF-1α, HOXA11-AS, C/EBPβ, and HOXA11-AS. First, the binding sequences for HIF-1α or C/EBPβ on HOXA11-AS were synthesized (WT) and mutated (Mut); after which, pGL3-promoter vectors (Thermo Fisher Scientific) containing the binding sites were constructed by GenePharma (Shanghai, China). Next, the vectors were transfected into WiT49 cells treated with control conditions or hypoxia. Finally, a Dual-Luciferase Reporter Assay System (Promega) was used to detect luciferase activity.